The NF-.kappa.B family of transcription factors are involved in the regulation of a wide variety of cellular responses. These transcription factors mediate extracellular signals that induce expression of genes involved in such diverse processes as cell division, inflammation, and apoptosis. See, for example, Baldwin, Annu. Rev. Immunol. 12, 141-179 (1996); Beg and Baltimore, Science 274, 782-274 (1996); Gilmore et al., Oncogene 13, 1267-1378 (1996); Mayo, et al, Science 278, 1812-1815 (1997); and Van Antwerp et al., Science 274, 787-789 (1996); Ashkenazi and Dixit, Science 281, 1305-1308 (1998).
NF-.kappa.B is anchored in the cytoplasm of most non-stimulated cells by a non-covalent interaction with one of several inhibitory proteins known as I.kappa.Bs. See for example, Baeuerle and Baltimore, Science 242, 540-546 (1988). Cellular stimuli associated with immune and inflammatory responses, for example inflammatory cytokines such as tumor necrosis factor .alpha. (TNF.alpha.) or interleukin-1 (IL-1), activate kinases, which in turn activate NF-.kappa.B by phosphorylating I.kappa.Bs. The kinases that phosphorylate I.kappa.Bs are called I.kappa.B kinases (IKKs).
Phosphorylation marks the I.kappa.Bs for ubiquitination and proteosome mediated degradation. The degradation and dissociation, of I.kappa.Bs from NF-.kappa.B unmasks the NF-.kappa.B nuclear localization signal, and facilitates the nuclear translocation of active NF-.kappa.B to the nucleus, thereby upregulating NF-.kappa.B responsive target genes. See, for example, Baeuerle and Henkel, Annu. Rev. Immunol. 12, 141-179 (1994); Baldwin, Annu.Rev. Immunol. 14, 649-683 (1996); Siebenlist et al., Annu.Rev.Cell Biol. 12, 405-455 (1994); and Verma et al, Genes Dev. 9, 2723-2735 (1995). Thus, this phosphorylation of I.kappa.Bs is a key regulatory step for NF-.kappa.B mediated processes.
The determination and characterization of kinases that directly phosphorylate I.kappa.Bs are instrumental in the delineation of signaling pathways involving NF-.kappa.B activation. Recently, an I.kappa.B kinase, designated IKK.alpha. but also referred to as CHUK (conserved helix-loop-helix ubiquitous kinase), was identified in a yeast-two-hybrid screen with NIK as bait. Regnier et al., Cell 90, 373-383 (1997). IKK.alpha. was determined to be responsible for the major I.kappa.B kinase activity induced by TNF stimulation of HeLa cells. DiDonato et al., Nature 388, 548-554 (1997). The identification of IKK.alpha. as a cytoplasmic kinase which phosphorylates I.kappa.B family members at their physiologically relevant sites and targets them for proteosome-mediated degradation was a major breakthrough.
The IKK.alpha. gene encodes a 745 amino-acid polypeptide (having a molecular mass of approximately 85 kDa). Murine and human IKK.alpha. cDNA clones were found to be almost identical. Connelly and Marcu, Cellular and Molecular Biology Research 41, 537-549 (1995).
Another kinase, termed IKK.beta., homologous to IKK.alpha., has also been reported. Stancovski and Baltimore, Cell 91, 299-302 (1997); Woronicz et al., Science 278, 866-869 (1997); and Zandi et al., Cell 91, 243-252 (1997). IKK.alpha. and IKK.beta. have 52% overall similarity to each other and 65% identity in the kinase domain. Zandi et al., Cell 91, 243-252 (1997). An I.kappa.B kinase termed T2K has also been described in U.S. Pat. No. 5,776,717 to Cao.
The known I.kappa.B protein kinases generally phosphorylate I.kappa.Bs at specific serine residues. For example, they specifically phosphorylate serines 32 and 36 of I.kappa.B.alpha.. Phosphorylation of both sites is required to efficiently target I.kappa.B.alpha. for destruction in vivo. Moreover, activation of IKK.alpha. and IKK.beta. occurs in response to NF-.kappa.B activating agents and mutant IKK.alpha. and IKK.beta. that are catalytically inactive block NF-.kappa.B stimulation by cytokines. These results highlight the important role played by I.kappa.B protein kinases in NF-.kappa.B activation processes. See Stancovski and Baltimore, Cell 91, 299-302 (1997) for a recent discussion of I.kappa.B kinases.
IKK.alpha. and IKK.beta. have structural motifs characteristic of the IKK kinases. This includes an amino terminal serine-threonine kinase domain separated from a carboxyl proximal helix-loop-helix (H-L-H) domain by a leucine zipper domain. These structural characteristics are unlike other kinases, and the domains are thought to be involved in protein-protein interactions.
Numerous proteins are involved in the signaling pathways that lead to immune, inflammatory, and apoptotic responses. A complete elucidation of these processes requires the identification of additional proteins that are involved and a determination of the protein interactions.
The discovery of additional proteins involved in these processes is important for controlling immune, apoptotic, and inflammatory processes. Thus, there is a great need for the identification and characterizion of additional proteins involved in IKK mediated cellular processes.